2-Methylene-(20S,25S)-19,26-Dinor-Vitamin D Analogs

ABSTRACT

This invention discloses 2-methylene-(20S,25S)-19,26-dinor-vitamin D analogs, and specifically 2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D 3 , and pharmaceutical uses therefor. This compound exhibits pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. This compound also has little, if any, calcemic activity and therefore may be used to treat autoimmune disorders or inflammatory diseases in humans as well as renal osteodystrophy. This compound may also be used for the treatment or prevention of obesity.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 61/017,228 filed on Dec. 28,2007, the content of which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to2-methylene-(20S,25S)-19,26-dinor-vitamin D analogs and theirpharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

SUMMARY OF THE INVENTION

The present invention is directed toward2-methylene-(20S,25S)-19,26-dinor-vitamin D analogs, their biologicalactivity, and various pharmaceutical uses for these compounds. These newvitamin D compounds not known heretofore are the 19-nor-vitamin Danalogs having a methylene group at the 2-position (C-2), a methyl groupat the 20-position (C-20) in its S-configuration, the replacement of themethyl group typically located at the 26 position (C-26) in the sidechain with a hydrogen atom, and the hydroxyl group at the 25-position(C-25) in its S-configuration. The preferred vitamin D analog is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃.

Structurally these 2-methylene-(20S,25S)-19,26-dinor-vitamin D analogsare characterized by the general formula I shown below:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group. The preferredanalog is 2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃which has the following formula Ia:

The above compounds I, particularly Ia, exhibit a desired, and highlyadvantageous, pattern of biological activity. These compounds arecharacterized by relatively high binding to vitamin D receptors. Thesecompounds have some ability to promote intestinal calcium transport invivo, in a dose dependent manner, but their ability would be classifiedas very low intestinal calcium transport activity, as compared to thatof 1α,25-dihydroxyvitamin D₃. These compounds I, and particularly Ia,also have very low ability to mobilize calcium from bone, as compared to1α,25-dihydroxyvitamin D₃. Hence, these compounds can be characterizedas having little, if any, calcemic activity. It is undesirable to raiseserum calcium to supraphysiologic levels when suppressing thepreproparathyroid hormone gene (Darwish & DeLuca, Arch. Biochem.Biophys. 365, 123-130, 1999) and parathyroid gland proliferation. Theseanalogs having little or no calcemic activity while very active ondifferentiation are expected to be useful as a therapy for suppressionof secondary hyperparathyroidism as well as renal osteodystrophy.

The compounds I, particularly Ia, of the invention have also beendiscovered to be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compounds of the invention.

The above compounds I, and particularly Ia, are also characterized byrelatively high cell differentiation activity. Thus, these compoundsalso provide a therapeutic agent for the treatment of psoriasis, or asan anti-cancer agent, especially against leukemia, colon cancer, breastcancer, skin cancer and prostate cancer. In addition, due to theirrelatively high cell differentiation activity, these compounds provide atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of these compounds thus not only results in moisturizingof skin but also improves the barrier function of skin.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of one or more of the compounds or thepharmaceutical compositions to the subject inhibits adipocytedifferentiation, inhibits gene transcription, and/or reduces body fat inthe animal subject.

One or more of the compounds may be present in a composition to treatthe above-noted diseases and disorders in an amount from about 0.01μg/gm to about 1000 μg/gm of the composition, preferably from about 0.1μg/gm to about 500 μg/gm of the composition, and may be administeredtopically, transdermally, orally, rectally, nasally, sublingually orparenterally in dosages of from about 0.01 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIGS. 1-5 illustrate various biological activities of2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃, hereinafterreferred to as “A4,” as compared to the native hormone1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of A4 and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of A4 and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to A4;

FIG. 4A is a bar graph illustrating the bone calcium mobilizationactivity of 1,25(OH)₂D₃ as compared to A4 in a first experiment using afirst group of animals;

FIG. 4B is a bar graph illustrating the bone calcium mobilizationactivity of 1,25(OH)₂D₃ as compared to A4 in a second experiment using asecond group of animals;

FIG. 5A is a bar graph illustrating the intestinal calcium transportactivity of 1,25(OH)₂D₃ as compared to A4 in a first experiment using afirst group of animals; and

FIG. 5B is a bar graph illustrating the intestinal calcium transportactivity of 1,25(OH)₂D₃ as compared to A4 in a second experiment using asecond group of animals.

DETAILED DESCRIPTION OF THE INVENTION

2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ (referred toherein as “A4”) a 19-nor vitamin D analog which is characterized by thepresence of a methylene substituent at the carbon 2 (C-2), a methylgroup at the 20-position (C-20) in its S-configuration, the replacementof the methyl group typically located at the 26-position (C-26) in theside chain with a hydrogen atom, and the hydroxyl group at the25-position (C-25) in its S-configuration, was synthesized and tested.Such vitamin D analog seemed an interesting target because therelatively small methylene group at the C-2 position should notinterfere with binding to the vitamin D receptor. Structurally, this19-nor analog is characterized by the general formula Ia previouslyillustrated herein, and its pro-drug (in protected hydroxy form) ischaracterized by general formula I previously illustrated herein.

The preparation of 2-methylene-(20S,25S)-19,26-dinor-vitamin D analogshaving the structure I can be accomplished by a common general method,i.e. the condensation of a bicyclic Windaus-Grundmann type ketone IIwith the allylic phosphine oxide III to the corresponding2-methylene-19,26-dinor-vitamin D analog IV followed by deprotection atC-1 and C-3 in the latter compound:

In the structures II, III and IV, groups X₁, X₂ and X₃ arehydroxy-protecting groups, preferably t-butyldimethylsilyl, it beingalso understood that any functionalities that might be sensitive, orthat interfere with the condensation reaction, be suitably protected asis well-known in the art. The process shown above represents anapplication of the convergent synthesis concept, which has been appliedeffectively for the preparation of vitamin D compounds [e.g. Lythgoe etal., J. Chem. Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev.9, 449 (1983); Toh et al., J. Org. Chem. 48, 1414 (1983); Baggiolini etal., J. Org. Chem. 51, 3098 (1986); Sardina et al., J. Org. Chem. 51,1264 (1986); J. Org. Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No.5,086,191; DeLuca et al., U.S. Pat. No. 5,536,713].

The hydrindanone of the general structure II is not known. It can beprepared by the method shown in Schemes 1, 2 and 3 herein (see thepreparation of compound A4).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compounds I and Ia isillustrated and described more completely in U.S. Pat. No. 5,843,928entitled “2-Alkylidene-19-Nor-Vitamin D Compounds” the specification ofwhich is specifically incorporated herein by reference.

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

More specifically, reference should be made to the followingillustrative example and description as well as to Schemes 1-3 hereinfor a detailed illustration of the preparation of compound A4.

In this example specific products identified by Arabic numerals (1, 2,3) refer to the specific structures so identified in the Schemes 1, 2and 3.

EXAMPLE

Chemistry. Ultraviolet (UV) absorption spectra were recorded with aHitachi Model 60-100 UV-vis spectrometer in the solvent noted. ¹Hnuclear magnetic resonance (NMR) spectra were recorded at 500 MHz with aBruker AM-500 FT spectrometer in deuteriochloroform. Chemical shifts (δ)are reported downfield from internal Me₄Si (δ 0.00). Mass spectra wererecorded at 70 eV on a Kratos DS-50 TC instrument equipped with a KratosMS-55 data system. Samples were introduced into the ion sourcemaintained at 120-250° C. via a direct insertion probe. High-performanceliquid chromatography (HPLC) was performed on a Waters Associates liquidchromatograph equipped with a Model 6000A solvent delivery system, aModel 6 UK Universal injector, a Model 486 tunable absorbance detector,and a differential R 401 refractometer.

Example 1 Preparation of(3S)-1-p-Toluenesulfonyloxy-3-triethylsilyloxy-butane (2)

To a stirred solution of the (S)-(+)-1,3-butanediol 1 (1 g, 11.1 mmol),DMAP (30 mg, 0.25 mmol) and Et₃N (4.6 mL, 3.33 g, 33 mmol) in anhydrousmethylene chloride (20 mL) p-toluenesulfonyl chloride (2.54 g, 13.3mmol) was added at 0° C. The reaction mixture was stirred at 4° C. for22 h. Methylene chloride was added and the mixture was washed withwater, dried (Na₂SO₄) and concentrated under reduced pressure. A residuewas chromatographed on silica gel with hexane/ethyl acetate (8:2, then1:1) to afford the tosylate (2.31 g, 85% yield) as a colorless oil.

To a stirred solution of the tosylate (2.31 g, 9.5 mmol) and2,6-lutidine (1.2 mL, 1.12 g, 10.5 mmol) in anhydrous methylene chloride(15 mL) triethylsilyl trifluoromethanesulfonate (2.1 mL, 2.51 g, 9.5mmol) was added at −50° C. The reaction mixture was allowed to warm toroom temperature (4 h) and stirring was continued for additional 20 h.Methylene chloride was added and the mixture was washed with water,dried (Na₂SO₄) and concentrated under reduced pressure. A residue waschromatographed on silica gel with hexane/ethyl acetate (97:3) to affordthe product 2 (2.71 g, 80% yield) as a colorless oil:

[α]_(D)+18.0 (c 2.38, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.77 (2H, d,J=8.2 Hz, o-H_(Ts)), 7.33 (2H, d, J=8.2 Hz, m-H_(Ts)), 4.10 (2H, t,J=6.1 Hz, 1-H₂), 3.90 (1H, m, 3-H), 2.43 (3H, s, Me_(Ts)), 1.72 (2H, m,2-H₂), 1.10 (3H, d, J=6.2 Hz, 4-H₃), 0.88 (9H, t, J=8.0 Hz, 3×SiCH₂CH ³), 0.50 (6H, q, J=8.0 Hz, 3×SiCH ² CH₃); ¹³C NMR (100 MHz) δ 144.62 (s,p-C_(Ts)), 133.03 (s, i-C_(Ts)), 129.72 (d, m-C_(Ts)), 127.82 (d,o-C_(Ts)), 67.78 (t, C-1), 64.46 (d, C-3), 38.47 (t, C-2), 23.82 (q,C-4), 21.52 (q, Me_(Ts)), 6.71 (q, SiCH₂ CH₃), 4.77 (t, SiCH₂CH₃); MS(EI) m/z 359 (5, MH⁺), 329 (87, M⁺-C₂H₅), 259 (100), 233 (54), 197 (50),179 (74), 163 (40), 149 (48), 135 (38), 115 (53), 91 (71); exact masscalculated for C₁₅H₂₅O₄SSi (M⁺-C₂H₅) 329.1243, found 329.1239.

Preparation of (3S)-1-Iodo-3-triethylsilyloxy-butane (3)

To a stirred solution of the tosylate 2 (2.71 g, 7.6 mmol) in anhydrousacetone (50 mL) potassium iodide (8 g, 48 mmol) was added and thereaction mixture was refluxed for 10 h. Water (30 mL) was added and thesolution was extracted with ethyl acetate. The combined organic phaseswere dried (Na₂SO₄) and concentrated under reduced pressure. The residuewas chromatographed on silica gel with hexane/ethyl acetate (97:3) togive the alcohol 3 (2.26 g, 95% yield) as a colorless oil:

[α]_(D)+36.3 (c 2.12, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 3.89 (1H, m,3-H), 3.22 (2H, t, J=7.0 Hz, 1-H₂), 1.91 (2H, m, 2-H₂), 1.16 (3H, d,J=6.1 Hz, 4-H₃), 0.96 (9H, t, J=7.9 Hz, 3×SiCH₂CH ³ ), 0.61 (6H, q,J=7.9 Hz, 3×SiCH ² CH₃); ¹³C NMR (100 MHz) δ 68.13 (d, C-3), 43.23 (t,C-2), 23.45 (q, C-4), 6.86 (q, SiCH₂ CH₃), 4.99 (t, SiCH₂CH₃), 3.34 (t,C-1); MS (EI) m/z 314 (1, M⁺), 299 (1, M⁺-CH₃), 285 (100, M⁺-C₂H₅), 257(97, M⁺-C₄H₉), 228 (51), 212 (98), 184 (58), 157 (62), 129 (33), 115(31); exact mass calculated for C₈H₁₈OISi (M⁺-C₂H₅) 285.0172, found285.0169.

Preparation of (3S)-Hydroxybutyl-triphenylphosphonium iodide (4)

To a stirred solution of the iodide 3 (1.67 g, 5.3 mmol) in acetonitrile(50 mL) triphenylphosphine (4.2 g, 16 mmol) was added and the reactionmixture was refluxed for 2 days. Acetonitrile was evaporated underreduced pressure, ethyl acetate (50 mL) was added and the mixture wasstirred at room temperature for 4 h. After removal of the solvent byfiltration the solid was washed with ethyl acetate, filtered off anddried. The pure phosphonium salt 4 (2.13 g, 87% yield) was obtained aswhite crystals:

¹H NMR (400 MHz, CD₃OD) δ 8.00-7.70 (15H, m, H_(Ph)), 3.89 (1H, m, 3-H),3.48 (2H, m, 1-H₂), 1.73 (2H, m, 2-H₂), 1.19 (3H, d, J=6.2 Hz, 4-H₃);¹³C NMR (100 MHz) δ 136.42 (d, p-C_(Ph)), 134.99 (d, J_(C-P)=10.1 Hz,m-C_(Ph)), 131.71 (d, J_(C-P)=13.1 Hz, o-C_(Ph)), 120.04 (s,J_(C-P)=86.5 Hz, i-C_(Ph)), 67.94 (d, J_(C-P)=16.2 Hz, C-3), 32.52 (t,J_(C-P)=4.1 Hz, C-2), 23.38 (q, C-4), 19.84 (t, J_(C-P)=53.7 Hz, C-1);exact mass calculated for C₂₂H₂₄OPI (M⁺) 335.1565, found 335.1571.

Preparation of (8S,20S)-des-A,B-20-(hydroxymethyl)pregnan-8-ol (5)

Ozone was passed through a solution of vitamin D₂ (3 g, 7.6 mmol) inmethanol (250 mL) and pyridine (2.44 g, 2.5 mL, 31 mmol) for 50 min at−78° C. The reaction mixture was then flushed with an oxygen for 15 minto remove the residual ozone and the solution was treated with NaBH₄(0.75 g, 20 mmol). After 20 min the second portion of NaBH₄ (0.75 g, 20mmol) was added and the mixture was allowed to warm to room temperature.The third portion of NaBH₄ (0.75 g, 20 mmol) was then added and thereaction mixture was stirred for 18 h. The reaction was quenched withwater (40 mL) and the solution was concentrated under reduced pressure.The residue was extracted with ethyl acetate and the combined organicphases were washed with 1M aq. HCl, saturated aq. NaHCO₃, dried (Na₂SO₄)and concentrated under reduced pressure. The residue was chromatographedon silica gel with hexane/ethyl acetate (75:25) to give the diol 5 (1.21g, 75% yield) as white crystals:

m.p. 106-108° C.; [α]_(D)+30.2° (c 1.46, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ 4.08 (1H, d, J=2.0 Hz, 8α-H), 3.63 (1H, dd, J=10.5, 3.1 Hz, 22-H),3.38 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.99 (1H, br.d, J=13.2 Hz), 1.03(3H, d, J=6.6 Hz, 21-H₃), 0.956 (3H, s, 18-H₃); ¹³C NMR (100 MHz) δ69.16 (d, C-8), 67.74 (t, C-22), 52.90 (d), 52.33 (d), 41.83 (s, C-13),40.19 (t), 38.20 (d), 33.53 (t), 26.62 (t), 22.54 (t), 17.36 (t), 16.59(q, C-21), 13.54 (q, C-18); MS (EI) m/z 212 (2, M⁺), 194 (34, M⁺-H₂O),179 (33, M⁺-H₂O—CH₃), 163 (18, M⁺-CH₂OH—H₂O), 135 (36), 125 (54), 111(100), 95 (63), 81 (67); exact mass calculated for C₁₃H₂₂O (M⁺-H₂O)194.1671, found 194.1665.

Preparation of (8S,20S)-des-A,B-8-benzoyloxy-20-(hydroxymethyl)pregnane(6)

Benzoyl chloride (2.4 g, 2 mL, 17 mmol) was added to a solution of thediol 5 (1.2 g, 5.7 mmol) and DMAP (30 mg, 0.2 mmol) in anhydrouspyridine (20 mL) at 0° C. The reaction mixture was stirred at 4° C. for24 h, diluted with methylene chloride (100 mL), washed with 5% aq. HCl,water, saturated aq. NaHCO₃, dried (Na₂SO₄) and concentrated underreduced pressure. The residue (3.39 g) was treated with a solution ofKOH (1 g, 15.5 mmol) in anhydrous ethanol (30 mL) at room temperature.After stirring of the reaction mixture for 3 h, ice and 5% aq. HCl wereadded until pH=6. The solution was extracted with ethyl acetate (3×50mL) and the combined organic phases were washed with saturated aq.NaHCO₃, dried (Na₂SO₄) and concentrated under reduced pressure. Theresidue was chromatographed on silica gel with hexane/ethyl acetate(75:25) to give the alcohol 6 (1.67 g, 93% yield) as a colorless oil:

[α]_(D)+56.0 (c 0.48, CHCl₃); ¹H NMR (400 MHz, CDCl₃+TMS) δ 8.08-8.02(2H, m, o-H_(Bz)), 7.59-7.53 (1H, m, p-H_(Bz)), 7.50-7.40 (2H, m,m-H_(Bz)), 5.42 (1H, d, J=2.4 Hz, 8α-H), 3.65 (1H, dd, J=10.5, 3.2 Hz,22-H), 3.39 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.08 (3H, d, J=5.3 Hz,21-H₃), 1.07 (3H, s, 18-H₃); ¹³C NMR (125 MHz) δ 166.70 (s, C═O), 132.93(d, p-C_(Bz)), 130.04 (s, i-C_(Bz)), 129.75 (d, o-C_(Bz)), 128.57 (d,m-C_(Bz)), 72.27 (d, C-8), 67.95 (t, C-22), 52.96 (d), 51.60 (d), 42.15(s, C-13), 39.98 (t), 38.61 (d), 30.73 (t), 26.81 (t), 22.91 (t), 18.20(t), 16.87 (q, C-21), 13.81 (q, C-18); MS (EI) m/z 316 (5, M⁺), 301 (3,M⁺-Me), 299 (1, M⁺-OH), 298 (2, M⁺-H₂O), 285 (10, M⁺-CH₂OH), 257 (6),230 (9), 194 (80), 135 (84), 105 (100); exact mass calculated forC₂₀H₂₈O₃ 316.2038, found 316.2019.

Preparation of (8S,20S)-des-A,B-8-benzoyloxy-20-formylpregnane (7)

Sulfur trioxide pyridine complex (1.94 g, 12.2 mmol) was added to asolution of the alcohol 6 (640 mg, 2.03 mmol), triethylamine (1.41 mL,1.02 g, 10.1 mmol) in anhydrous methylene chloride (10 mL) and anhydrousDMSO (2 mL) at 0° C. The reaction mixture was stirred under argon at 0°C. for 1 h and then concentrated. The residue was diluted with ethylacetate, washed with brine, dried (Na₂SO₄) and concentrated. The residuewas purified by column chromatography on silica gel with hexane/ethylacetate (95:5) to give the aldehyde 7 (529 mg, 83% yield) as an oil:

1H NMR (400 MHz, CDCl₃+TMS) δ 9.60 (1H, d, J=3.1 Hz, CHO), 8.05 (2H, m,o-H_(Bz)), 7.57 (1H, m, p-H_(Bz)), 7.45 (2H, m, m-H_(Bz)), 5.44 (1H, s,8α-H), 2.39 (1H, m, 20-H), 2.03 (2H, dm, J=11.5 Hz), 1.15 (3H, d, J=6.9Hz, 21-H₃), 1.10 (3H, s, 18-H₃); ¹³C NMR (100 MHz) δ 204.78 (d, CHO),166.70 (s, C═O), 132.78 (d, p-Bz), 130.69 (s, i-Bz), 129.50 (d, o-Bz),128.38, (d, m-Bz), 71.66 (d, C-8), 51.30 (d), 50.95 (d), 49.20 (d),42.38 (s, C-13), 39.62 (t), 30.47 (t), 25.99 (t), 22.92 (t), 17.92 (t),13.90 (q), 13.35 (q); MS (EI) m/z 314 (1, M⁺), 299 (0.5, M⁺-Me), 286 (1,M⁺-CO), 285 (5, M⁺-CHO), 257 (1, M⁺-C₃H₅O), 209 (10, M⁺-PhCO), 192 (38),134 (60), 105 (100), 77 (50); exact mass calculated for C₂₀H₂₆O₃314.1882, found 314.1887.

Preparation of (8S,20R)-des-A,B-8-benzoyloxy-20-(hydroxymethyl)pregnane(8)

The aldehyde 7 (364 mg, 1.12 mmol) was dissolved in methylene chloride(15 mL) and a 40% aq. n-Bu₄NOH solution (1.47 mL, 1.45 g, 2.24 mmol) wasadded. The resulting mixture was stirred under argon at room temperaturefor 16 h, diluted with methylene chloride (20 mL), washed with water,dried (Na₂SO₄) and concentrated under reduced pressure. A residue waschromatographed on silica gel with hexane/ethyl acetate (95:5) to afforda mixture of aldehyde 7 and its 20-epimer (292 mg, 80% yield) in ca. 1:2ratio (by ¹H NMR).

This mixture of aldehydes (292 mg, 0.9 mmol) was dissolved in THF (5 mL)and NaBH₄ (64 mg, 1.7 mmol) was added, followed by a dropwise additionof ethanol (5 mL). The reaction mixture was stirred at room temperaturefor 30 min and it was quenched with a saturated aq. NH₄Cl solution. Themixture was extracted with ether (3×20 mL) and the combined organicphase was washed with water, dried (Na₂SO₄) and concentrated underreduced pressure. The residue was chromatographed on silica gel withhexane/ethyl acetate (96:4→80:20) to give the desired, pure(20R)-alcohol 8 (160 mg, 55% yield) as an oil and a mixture of 8 and its20-epimer 6 (126 mg, 43% yield) in ca. 1:3 ratio (by ¹H NMR).

[α]_(D)+50.1 (c 1.09, CHCl₃); ¹H NMR (400 MHz, CDCl₃+TMS) δ 8.05 (2H, m,o-H_(Bz)), 7.55 (1H, m, p-H_(Bz)), 7.44 (2H, m, m-H_(Bz)), 5.41 (1H, s,8α-H), 3.77 (1H, dd, J=10.4, 3.3 Hz, 22-H), 3.45 (1H, dd, J=10.4, 7.4Hz, 22-H), 1.067 (3H, s, 18-H₃), 0.973 (3H, d, J=6.6 Hz, 21-H₃); ¹³C NMR(100 MHz) δ 166.36 (s, C═O), 132.61 (d, p-C_(Bz)), 130.63 (s, i-C_(Bz)),129.39 (d, o-C_(Bz)), 128.23 (d, m-C_(Bz)), 71.97 (d, C-8), 66.42 (t,C-22), 52.65 (d), 51.38 (d), 41.58 (s, C-13), 39.16 (t), 37.45 (d),30.38 (t), 26.29 (t), 22.35 (t), 17.89 (t), 16.42 (q, C-21), 13.78 (q,C-18); MS (EI) m/z 316 (16, M⁺), 301 (5, M⁺-Me), 299 (2, M⁺-OH), 298 (3,M⁺-H₂O), 285 (9, M⁺-CH₂OH), 257 (5), 242 (11), 230 (8), 194 (60), 147(71), 105 (100); exact mass calculated for C₂₀H₂₈O₃ 316.2038, found316.2050.

Preparation of (8S,20R)-des-A,B-8-benzoyloxy-20-formylpregnane (9)

Sulfur trioxide pyridine complex (258 mg, 1.62 mmol) was added to asolution of the alcohol 8 (85 mg, 0.27 mmol), triethylamine (188 μL, 136mg, 1.35 mmol) in anhydrous methylene chloride (5 mL) and anhydrous DMSO(1 mL) at 0° C. The reaction mixture was stirred under argon at 0° C.for 1 h and then concentrated. The residue was diluted with ethylacetate, washed with brine, dried (Na₂SO₄) and concentrated. The residuewas purified by column chromatography on silica gel with hexane/ethylacetate (95:5) to give the aldehyde 9 (70 mg, 83% yield) as an oil:

[α]_(D)+28.8 (c 0.88, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 9.55 (1H, d,J=5.0 Hz, CHO), 8.02 (2H, m, o-H_(Bz)), 7.54 (1H, m, p-H_(Bz)), 7.43(2H, m, m-H_(Bz)), 5.42 (1H, s, 8α-H), 2.35 (1H, m, 20-H), 2.07 (1H, m),1.87 (1H, m), 1.05 (3H, s, 18-H₃), 1.04 (3H, d, J=7.8 Hz, 21-H₃); ¹³CNMR (125 MHz) δ 205.51 (d, CHO), 166.34 (s, C═O), 132.76 (d, p-C_(Bz)),130.62 (s, i-C_(Bz)), 129.47 (d, o-C_(Bz)), 128.35, (d, m-C_(Bz)), 71.52(d, C-8), 52.08 (d), 51.08 (d), 48.40 (d), 41.55 (s, C-13), 38.54 (t),30.41 (t), 25.28 (t), 22.08 (t), 17.68 (t), 14.49 (q), 13.38 (q); MS(EI) m/z 314 (2, M⁺), 285 (3, M⁺-CHO), 209 (8, M⁺-PhCO), 192 (30,M⁺-PhCOOH), 177 (14), 134 (45), 105 (100), 77 (50); exact masscalculated for C₁₉H₂₅O₂ (M⁺-CHO) 285.1855, found 285.1849.

Preparation of(8S,20S)-des-A,B-8-benzoyloxy-20-[(4S)-hydroxy-pent-(1E)-en-yl]pregnane(10)

To a stirred suspension of the phosphonium salt 4 (201 mg, 0.6 mmol) inanhydrous THF (5 mL) butyllithium (1.6 M, 560 μL, 0.9 mmol) was added at−20° C. The solution turned deep orange. After 1 h a precooled (−20° C.)solution of the aldehyde 9 (65 mg, 0.2 mmol) in anhydrous THF (2 mL) wasadded and the reaction mixture was stirred at −20° C. for 3 h and atroom temperature for 18 h. The reaction was quenched with water and themixture was extracted with ethyl acetate. Combined organic phases werewashed with brine, dried (Na₂SO₄) and evaporated. The residue waschromatographed on silica gel with hexane/ethyl acetate (95:5) to givethe product 10 (37 mg, 50% yield):

[α]_(D)−11.4 (c 1.4, CHCl₃); ¹H NMR (400 MHz, CDCl₃+TMS) δ 8.05 (2H, m,o-H_(Bz)), 7.55 (1H, m, p-H_(Bz)), 7.44 (2H, m, m-H_(Bz)), 5.41 (1H, s,8α-H), 5.45-5.25 (2H, m, 22-H and 23-H), 3.81 (1H, m, 25-H), 1.20 (3H,d, J=6.1 Hz, 27-H₃), 1.04 (3H, s, 18-H₃), 0.94 (3H, d, J=6.6 Hz, 21-H₃);¹³C NMR (100 MHz) δ 166.45 (s, C═O), 141.11 (d, C-22), 132.66 (d,p-C_(Bz)), 130.87 (s, i-C_(Bz)), 129.53 (d, o-C_(Bz)), 128.32 (d,m-C_(Bz)), 123.41 (d, C-23), 72.09 (d, C-8), 67.23 (d, C-25), 56.34 (d),51.47 (d), 42.56 (t), 41.95 (s, C-13), 40.15 (d), 39.37 (t), 30.59 (t),26.80 (t), 22.73 (q, C-27), 22.49 (t), 21.56 (q, C-21), 17.83 (t), 13.85(q, C-18); MS (EI) m/z 370 (8, M⁺), 355 (0.5, M⁺-CH₃), 326 (2,M⁺-C₂H₄O), 284 (12, M⁺-C₅H₁₀O), 265 (2, M⁺-PhCO), 248 (28, M⁺-PhCOOH),230 (9), 204 (17), 189 (10), 162 (63), 135 (71), 105 (100); exact masscalculated for C₂₄H₃₄O₃Na (MNa⁺) 393.2406, found 393.2410.

Preparation of(8S,20S)-des-A,B-8-benzoyloxy-20-[(4S)-hydroxy-pentyl]pregnane (11)

A solution of the compound 10 (37 mg, 0.1 mmol) in methanol (6 mL) washydrogenated for 17 h in the presence of 10% palladium on powderedcharcoal (6 mg). The reaction mixture was filtered through a bed ofCelite with several methanol washes, the filtrate was concentrated andthe residue was chromatographed on silica gel with hexane/ethyl acetate(95:5) to give the product 11 (24 mg, 65% yield):

[α]_(D)+32.6 (c 0.9, CHCl₃); ¹H NMR (400 MHz, CDCl₃+TMS) δ 8.06 (2H, m,o-H_(Bz)), 7.56 (1H, m, p-H_(Bz)), 7.45 (2H, m, m-H_(Bz)), 5.41 (1H, s,8α-H), 3.81 (1H, m, 25-H), 2.02 (2H, m), 1.83 (2H, m), 1.20 (3H, d,J=6.1 Hz, 27-H₃), 1.05 (3H, s, 18-H₃), 0.85 (3H, d, J=6.6 Hz, 21-H₃);¹³C NMR (100 MHz) δ 166.47 (s, C═O), 132.66 (d, p-C_(Bz)), 130.86 (s,i-C_(Bz)), 129.53 (d, o-C_(Bz)), 128.32 (d, m-C_(Bz)), 72.23 (d, C-8),68.25 (d, C-25), 55.96 (d), 51.64 (d), 41.95 (s, C-13), 39.85 (t), 39.71(t), 35.24 (t), 34.84 (d), 30.54 (t), 26.94 (t), 23.51 (q, C-27), 22.52(t), 22.39 (t), 18.47 (q, C-21), 18.06 (t), 13.83 (q, C-18); MS (EI) m/z372 (8, M⁺), 354 (2, M⁺-H₂O), 327 (0.5, M⁺-C₂H₅O), 285 (1, M⁺-C₅H₁₁O),267 (4, M⁺-PhCO), 250 (59, M⁺-PhCOOH), 232 (18), 163 (23), 135 (64), 105(100); exact mass calculated for C₂₄H₃₆O₃Na (MNa⁺) 395.2562, found395.2558.

Preparation of(8S,20S)-des-A,B-8-benzoyloxy-20-[(4S)-tert-butyldimethylsilyloxy-pentyl]pregnane(12)

tert-Butyldimethylsilyl trifluoromethanesulfonate (30 μL, 34 mg, 0.13mmol) was added to a solution of the alcohol 11 (24 mg, 65 μmol) and2,6-lutidine (30 μL, 28 mg, 0.26 mmol) in anhydrous methylene chloride(3 mL) at −20° C. The mixture was stirred under argon at 0° C. for 1 h.The reaction was quenched with water and extracted with methylenechloride. The combined organic phases were washed with brine, dried(Na₂SO₄) and concentrated under reduced pressure. The residue waschromatographed on silica gel with hexane and hexane/ethyl acetate(97:3) to give the product 12 (32 mg, 100%):

[α]_(D)+25.0 (c 0.55, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.06 (2H, m,o-H_(Bz)), 7.56 (1H, m, p-H_(Bz)), 7.45 (2H, m, m-H_(Bz)), 5.41 (1H, s,8α-H), 3.77 (1H, m, 25-H), 2.02 (2H, m), 1.82 (2H, m), 1.13 (3H, d,J=6.0 Hz, 27-H₃), 1.04 (3H, s, 18-H₃), 0.90 (9H, s, Si-t-Bu), 0.83 (3H,d, J=6.5 Hz, 21-H₃), 0.06 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 166.50 (s,C═O), 132.66 (d, p-C_(Bz)), 130.91 (s, i-C_(Bz)), 129.55 (d, o-C_(Bz)),128.33 (d, m-C_(Bz)), 72.27 (d, C-8), 68.81 (d, C-25), 55.99 (d), 51.67(d), 41.96 (s, C-13), 40.21 (t), 39.84 (t), 35.37 (t), 34.86 (d), 30.58(t), 26.95 (t), 25.92 (q, SiCMe ³ ), 23.88 (q, C-27), 22.55 (t), 22.46(t), 18.48 (q, C-21), 18.17 (s, SiCMe₃), 18.05 (t), 13.79 (q, C-18),−4.39 (q, SiMe), −4.68 (q, SiMe); MS (EI) m/z 485 (1, M⁺-H), 471 (1,M⁺-CH₃), 307 (9, M⁺-PhCOOH—C₄H₉), 233 (71, M⁺-PhCOOH-t-BuSiMe₂O), 197(71), 179 (92), 163 (81), 135 (71), 105 (100); exact mass calculated forC₃₀H₅₀O₃SiNa (MNa⁺) 509.3427, found 509.3446.

Preparation of(8S,20S)-des-A,B-20-[(4S)-tert-butyldimethylsilyloxy-pentyl]pregnan-8-ol(13)

A solution of sodium hydroxide in ethanol (2.5M, 2 mL) was added to astirred solution of the benzoate 12 (31 mg, 64 μmol) in anhydrousethanol (10 mL) and the reaction mixture was refluxed for 18 h. Themixture was cooled to room temperature, neutralized with 5% aq. HCl andextracted with dichloromethane. Combined organic phases were washed withsaturated aq. NaHCO₃, dried (Na₂SO₄) and evaporated. The residue waschromatographed on silica gel with hexane/ethyl acetate (95:5) to givethe alcohol 13 (18 mg, 74% yield):

[α]_(D)+14.3 (c 0.8, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.07 (1H, d,J=2.3 Hz, 8α-H), 3.75 (1H, m, 25-H), 1.97 (1H, m), 1.80 (3H, m), 1.11(3H, d, J=6.1 Hz, 27-H₃), 0.92 (3H, s, 18-H₃), 0.88 (9H, s, Si-t-Bu),0.81 (3H, d, J=6.6 Hz, 21-H₃), 0.04 (6H, s, SiMe₂); ¹³C NMR (125 MHz) δ69.44 (d, C-8), 68.80 (d, C-25), 56.21 (d), 52.65 (d), 41.88 (s, C-13),40.29 (t), 40.20 (t), 35.29 (t), 34.76 (d), 33.57 (t), 27.07 (t), 25.91(q, SiCMe ³ ), 23.88 (q, C-27), 22.46 (t), 22.41 (t), 18.49 (q, C-21),18.17 (s, SiCMe₃), 17.45 (t), 13.76 (q, C-18), −4.40 (q, SiMe), −4.70(q, SiMe); MS (EI) m/z 382 (2, M⁺), 367 (4, M⁺-CH₃), 325 (8, M⁺-C₄H₉),307 (3, M⁺-C₄H₉—H₂O), 233 (73), 191 (53), 177 (89), 163 (86) 149 (66),135 (98), 123 (75), 109 (93), 97 (100); exact mass calculated forC₁₉H₃₇O₂Si (M⁺-C₄H₉) 325.2563, found 325.2567.

Preparation of(20S)-des-A,B-20-[(4S)-tert-butyldimethylsilyloxy-pentyl]pregnan-8-one(14)

Molecular sieves A4 (50 mg) were added to a solution of4-methylmorpholine (17 mg, 0.17 mmol) in dichloromethane (0.5 mL). Themixture was stirred at room temperature for 15 min andtetrapropylammonium perruthenate (2 mg, 6 μmol) was added, followed by asolution of alcohol 13 (18 mg, 47 μmol) in dichloromethane (300+300 μL).The resulting suspension was stirred at room temperature for 1 h. Thereaction mixture was filtered through a Waters silica Sep-Pak cartridge(5 g) that was further washed with dichloromethane. After removal of thesolvent the ketone 14 (17 mg, 95% yield) was obtained as a colorlessoil:

[α]_(D)−20.2 (c 0.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 3.76 (1H, m,25-H), 2.44 (1H, dd, J=11.4, 7.7 Hz), 1.12 (3H, d, J=6.1 Hz, 27-H₃),0.89 (9H, s, Si-t-Bu), 0.84 (3H, d, J=5.9 Hz, 21-H₃), 0.63 (3H, s,18-H₃), 0.05 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 212.12 (s), 68.73 (d,C-25), 62.02 (d), 56.18 (d), 49.93 (s, C-13), 40.96 (t), 40.14 (t),38.85 (t), 35.54 (t), 34.86 (d), 27.16 (t), 25.90 (q, SiCMe ³ ), 24.03(t), 23.89 (q, C-27), 22.42 (t), 18.93 (t), 18.44 (q, C-21), 18.15 (s,SiCMe₃), 12.70 (q, C-18), −4.38 (q, SiMe), −4.69 (q, SiMe); MS (EI) m/z380 (2, M⁺), 379 (3, M⁺-H), 365 (14, M⁺-CH₃), 324 (60, M⁺-C₄H₈), 267(17), 253 (28), 231 (59), 189 (61), 161 (54), 135 (76), 95 (90), 75(100); exact mass calculated for C₁₉H₃₅O₂Si (M⁺-C₄H₉) 323.2406, found323.2405.

Preparation of (20S,25S)-2-Methylene-19,26-dinor-1α,25-dihydroxyvitaminD₃ (17)

To a solution of phosphine oxide 15 (74 mg, 127 μmol) in anhydrous THF(500 μL) at −20° C. was slowly added PhLi (1.8 M in di-n-butylether, 105μL, 189 μmol) under argon with stirring. The solution turned deeporange. After 30 min the mixture was cooled to −78° C. and a precooled(−78° C.) solution of ketone 14 (16 mg, 42 μmol) in anhydrous THF(200+100 μL) was slowly added. The mixture was stirred under argon at−78° C. for 3 h and at 0° C. for 18 h. Ethyl acetate was added, and theorganic phase was washed with brine, dried (Na₂SO₄) and evaporated. Theresidue was dissolved in hexane and applied on a Waters silica Sep-Pakcartridge (2 g). The cartridge was washed with hexane and hexane/ethylacetate (99.5:0.5) to give 19-norvitamin derivative 16 (25 mg, 80%yield). Then the Sep-Pak was washed with ethyl acetate to recoverdiphenylphosphine oxide 15 (40 mg). For analytical purpose a sample ofthe protected vitamin 16 was further purified by HPLC (9.4×250 mm ZorbaxSil column, 4 mL/min, hexane/2-propanol (99.9:0.1) solvent system,R_(t)=3.51 min):

UV (in hexane) λ_(max) 262.6, 253.2, 244.8 nm; ¹H NMR (400 MHz, CDCl₃) δ6.23 and 5.85 (each 1H, each d, J=11.1 Hz, 6- and 7-H), 4.98 and 4.93(each 1H, each s, ═CH₂), 4.42 (2H, m, 1β- and 3α-H), 3.77 (1H, m, 25-H),2.83 (1H, dm, J=11.8 Hz, 9β-H), 2.52 (1H, dd, J=13.3, 5.9 Hz, 10α-H),2.47 (1H, dd, J=12.4, 4.3 Hz, 4α-H), 2.33 (1H, dm, J=13.3 Hz, 10β-H),2.19 (1H, dd, J=12.4, 8.5 Hz, 4β-H), 1.12 (3H, d, J=6.0 Hz, 27-H₃),0.903 (9H, s, Si-t-Bu), 0.897 (9H, s, Si-t-Bu), 0.871 (9H, s, Si-t-Bu),0.84 (3H, d, J=6.5 Hz, 21-H₃), 0.547 (3H, s, 18-H₃), 0.086 (3H, s,SiMe), 0.072 (3H, s, SiMe), 0.055 (9H, s, 3×SiMe), 0.032 (3H, s, SiMe);¹³C NMR (100 MHz) δ 152.98 (s, C-2), 141.24 (s, C-8), 132.70 (s, C-5),122.42 (d, C-6), 116.09 (d, C-7), 106.25 (t, ═CH₂), 72.52 and 71.63(each d, C-1 and C-3), 68.80 (d, C-25), 56.32 (d), 56.17 (d), 47.60 (t),45.70 (s, C-13), 40.50 (t), 40.19 (t), 38.55 (t), 35.60 (t), 35.52 (d),28.76 (t), 27.42 (t), 25.92 (q, SiCMe ³ ), 25.84 (q, SiCMe ³ ), 25.78(q, SiCMe₃ ), 23.87 (q, C-27), 23.43 (t), 22.55 (t), 22.10 (t), 18.55(q, C-21), 18.25 (s, SiCMe₃), 18.17 (s, 2×SiCMe₃), 12.30 (q, C-18),−4.39 (q, SiMe), −4.69 (q, SiMe), −4.86 (q, 2×SiMe), −4.91 (q, SiMe),−5.10 (q, SiMe); exact mass calculated for C₄₄H₈₄O₃Si₃Na (MNa⁺)767.5626, found 767.5621.

The protected vitamin 16 (25 mg, 34 μmol) was dissolved in THF (2 mL)and acetonitrile (2 mL). A solution of aq. 48% HF in acetonitrile (1:9ratio, 2 mL) was added at 0° C. and the resulting mixture was stirred atroom temperature for 6 h. Saturated aq. NaHCO₃ solution was added andthe reaction mixture was extracted with ethyl acetate. The combinedorganic phases were washed with brine, dried (Na₂SO₄) and concentratedunder reduced pressure. The residue was diluted with 2 mL ofhexane/ethyl acetate (8:2) and applied on a Waters silica Sep-Pakcartridge (2 g). An elution with hexane/ethyl acetate (8:2) and laterwith ethyl acetate gave the crude product 17 (14 mg). The vitamin 17 wasfurther purified by reverse phase HPLC [9.4×250 mm Zorbax EclipseXDB-C18 column, 3 mL/min, methanol/water (85:15) solvent system,R_(t)=10.67 min.] to give a colorless oil (11.34 mg, 83% yield):

UV (in EtOH) λ_(max) 261.4, 252.2, 244.4 nm; ¹H NMR (500 MHz, CDCl₃) δ6.35 and 5.88 (1H and 1H, each d, J=11.2 Hz, 6- and 7-H), 5.10 and 5.08(each 1H, each s, ═CH₂), 4.47 (2H, m, 1β- and 3α-H), 3.78 (1H, m, 25-H),2.84 (1H, dd, J=13.1, 4.4 Hz, 10β-H), 2.81 (1H, br d, J=11.9 Hz, 9β-H),2.56 (1H, dd, J=13.4, 3.6 Hz, 4α-H), 2.32 (1H, dd, J=13.4, 6.1 Hz,4β-H), 2.28 (1H, dd, J=13.1, 8.4 Hz, 10α-H), 1.18 (3H, d, J=6.2 Hz,27-H₃), 0.84 (3H, d, J=6.5 Hz, 21-H₃), 0.543 (3H, s, 18-H₃); ¹³C NMR(125 MHz) δ 151.98 (s, C-2), 143.35 (s, C-8), 130.43 (s, C-5), 124.22(d, C-6), 115.31 (d, C-7), 107.70 (t, ═CH₂), 71.79 and 70.66 (each d,C-1 and C-3), 68.29 (d, C-25), 56.33 (d), 56.13 (d), 45.80 (t), 45.80(s, C-13), 40.34 (t), 39.74 (t), 38.14 (t), 35.55 (t), 35.41 (d), 28.94(t), 27.28 (t), 23.48 (t), 23.48 (q, C-27), 22.43 (t), 22.14 (t), 18.52(q, C-21), 12.36 (q, C-18); MS (EI) m/z 402 (100, M⁺), 384 (3, M⁺-H₂O),369 (2, M⁺-H₂O—CH₃), 351 (2, M⁺-2H₂O—CH₃), 287 (6, M⁺-C₇H₁₅O), 269 (14),251 (15), 192 (12), 161 (16), 147 (48), 135 (69), 95 (68); exact masscalculated for C₂₆H₄₂O₃ (M⁺) 402.3134, found 402.3147.

BIOLOGICAL ACTIVITY OF2-METHYLENE-(20S,25S)-19,26-DINOR-1α,25-DIHYDROXYVITAMIN D₃

The introduction of a methylene group to the 2-position, a methyl groupat the 20-position (C-20) in its S-configuration, the replacement of themethyl group typically located at the 26 position (C-26) in the sidechain with a hydrogen atom, and the hydroxyl group at the 25-position(C-25) in its S-configuration, had little effect on binding of A4 to thefull length recombinant rat vitamin D receptor, as compared to1α,25-dihydroxyvitamin D₃. The compound A4 bound with nearly the sameaffinity to the nuclear vitamin D receptor as compared to the standard1,25-(OH)₂D₃ (FIG. 1). It might be expected from these results thatcompound A4 would have equivalent biological activity. Surprisingly,however, compound A4 is a highly selective analog with unique biologicalactivity.

FIGS. 5A and 5B show that A4 has some ability to increase intestinalcalcium transport activity in vivo, in a dose dependent manner, but itclearly has very little activity as compared to that of1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport. In two batches of rats, A4failed to stimulate intestinal calcium transport as potently as1,25(OH)₂D₃.

FIGS. 4A and 4B demonstrate that A4 has very little bone calciummobilization activity, as compared to 1,25(OH)₂D₃. Two separate studiesclearly show that A4 has significantly less bone calcium mobilizationactivity compared to 1,25(OH)₂D₃. A4 is approximately 100 times lesspotent than the native hormone in mobilizing bone calcium store becauseit required at least 100 times more A4 to cause the same increase inserum calcium.

FIGS. 4 and 5 thus illustrate that A4 may be characterized as havinglittle, if any, calcemic activity.

FIG. 2 illustrates that A4 is about 10 times more potent than1,25(OH)₂D₃ on HL-60 cell differentiation, i.e. causing thedifferentiation of HL-60 cells into monocytes, making it an excellentcandidate for the treatment of psoriasis and cancer, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer.In addition, due to its relatively high cell differentiation activity,this compound provides a therapeutic agent for the treatment of variousskin conditions including wrinkles, lack of adequate dermal hydration,i.e. dry skin, lack of adequate skin firmness, i.e. slack skin, andinsufficient sebum secretion. Use of this compound thus not only resultsin moisturizing of skin but also improves the barrier function of skin.

FIG. 3 illustrates that in bone cells the compound A4 is one log, i.e.10 times, more potent than 1,25(OH)₂D₃ in increasing transcription ofthe 24-hydroxylase gene. This result, together with the celldifferentiation activity of FIG. 2, suggests that A4 will be veryeffective in psoriasis because it has direct cellular activity incausing cell differentiation, gene transcription, and in suppressingcell growth. These data also indicate that A4 may have significantactivity as an anti-cancer agent, especially against leukemia, coloncancer, breast cancer, skin cancer and prostate cancer.

The strong activity of A4 on HL-60 differentiation suggests it will beactive in suppressing growth of parathyroid glands and in thesuppression of the preproparathyroid gene.

Experimental Methods

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wereoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.

RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (0.47% Ca)diet +AEK oil for one week followed by Diet 11 (0.02% Ca) +AEK oil for 3weeks. The rats were then switched to a diet containing 0.47% Ca for oneweek followed by two weeks on a diet containing 0.02% Ca. Doseadministration began during the last week on 0.02% calcium diet. Fourconsecutive ip doses were given approximately 24 hours apart.Twenty-four hours after the last dose, blood was collected from thesevered neck and the concentration of serum calcium determined as ameasure of bone calcium mobilization. The first 10 cm of the intestinewas also collected for intestinal calcium transport analysis using theeverted gut sac method.

Interpretation of Data

Summary of Biological Findings. This compound binds the VDR with nearlythe same affinity as the native hormone, but displays approximately 10times greater cell differentiation activity and more than 10 times invitro gene transcription activity compared to 1,25(OH)₂D₃. In vivo thiscompound exhibits significantly less bone calcium mobilization andintestinal calcium transport activities compared to the native hormonemaking this compound a potentially valuable agent for the treatment ofsuch diseases as cancer, secondary hyperparathyroidism, renalosteodystrophy, autoimmune diseases, skin conditions, and psoriasis.

VDR binding, HL60 cell differentiation, and transcription activity. A4(K_(i)=6×10⁻¹¹M) is equally as active as the natural hormone1α,25-dihydroxyvitamin D₃ (K_(i)=6×10⁻¹¹M) in its ability to competewith [³H]-1,25(OH)₂D₃ for binding to the full-length recombinant ratvitamin D receptor (FIG. 1). A4 displays about 10 times greater activity(EC₅₀=5×10⁻¹⁰M) in its ability (efficacy or potency) to promote HL-60cell differentiation as compared to 1α,25-dihydroxyvitamin D₃(EC₅₀=2×10⁻⁹M) (See FIG. 2). Also, compound A4 (EC₅₀=3×10⁻¹¹M) has morethan 10 times greater transcriptional activity in bone cells than1α,25-dihydroxyvitamin D₃ (EC₅₀=2×10⁻¹⁰M) (See FIG. 3). These resultssuggest that A4 will be very effective in psoriasis because it hasdirect cellular activity in causing cell differentiation, genetranscription, and in suppressing cell growth. These data also indicatethat A4 will have significant activity as an anti-cancer agent,especially against leukemia, colon cancer, breast cancer, skin cancerand prostate cancer, as well as against skin conditions such as dry skin(lack of dermal hydration), undue skin slackness (insufficient skinfirmness), insufficient sebum secretion and wrinkles. It would also beexpected to be very active in suppressing secondary hyperparathyroidism,especially in subjects having chronic kidney disease and subjects ondialysis.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of A4 and 1,25(OH)₂D₃ in intestineand bone were tested. As expected, the native hormone (1,25(OH)₂D₃)increased serum calcium levels at all dosages (FIGS. 4A and 4B). The twoseparate studies reported in FIGS. 4A and 4B show that A4 has little, ifany, activity in mobilizing calcium from bone. Administration of A4 at260 pmol/day for 4 consecutive days did not result in mobilization ofbone calcium, and increasing the amount of A4 to 7020 pmol/day was alsowithout any substantial effect.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIGS. 5A and 5B). These results showthat the compound A4 promotes intestinal calcium transport only slightlywhen administered at 260 pmol/day, whereas 1,25(OH)₂D₃ promotes asignificant increase at the 260 pmol/day dose. It was only when 7020pmol/day of A4 was administered that significant intestinal calciumtransport activity was recorded, an almost 30-fold increase in dosageover the 260 pmol/day dose. Thus, it may be concluded that A4 isessentially devoid of intestinal calcium transport activity at therecommended lower doses.

These results illustrate that A4 is an excellent candidate for numeroushuman therapies as described herein, and that it may be particularlyuseful in a number of circumstances such as suppression of secondaryhyperparathyroidism of renal osteodystrophy, autoimmune diseases,cancer, numerous types of skin conditions, and psoriasis. A4 is anexcellent candidate for treating psoriasis because: (1) it hassignificant VDR binding, transcription activity and cellulardifferentiation activity; (2) it is devoid of hypercalcemic liability atrelatively low doses, unlike 1,25(OH)₂D₃; and (3) it is easilysynthesized. Since A4 has significant binding activity to the vitamin Dreceptor, but has little ability to raise blood serum calcium, it mayalso be particularly useful for the treatment of secondaryhyperparathyroidism, especially in subjects diagnosed with chronickidney disease and subjects on dialysis, as well as the treatment ofrenal osteodystrophy.

These data also indicate that the compound A4 of the invention may beespecially suited for treatment and prophylaxis of human disorders whichare characterized by an imbalance in the immune system, e.g. inautoimmune diseases, including multiple sclerosis, lupus, diabetesmellitus, host versus graft rejection, and rejection of organtransplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compound A4 of the invention.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of the compound or the pharmaceuticalcompositions to the subject inhibits adipocyte differentiation, inhibitsgene transcription, and/or reduces body fat in the animal subject. Theanimal may be a human, a domestic animal such as a dog or a cat, or anagricultural animal, especially those that provide meat for humanconsumption, such as fowl like chickens, turkeys, pheasant or quail, aswell as bovine, ovine, caprine, or porcine animals.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I may be formulated for pharmaceuticalapplications as a solution in innocuous solvents, or as an emulsion,suspension or dispersion in suitable solvents or carriers, or as pills,tablets or capsules, together with solid carriers, according toconventional methods known in the art. Any such formulations may alsocontain other pharmaceutically-acceptable and non-toxic excipients suchas stabilizers, anti-oxidants, binders, coloring agents or emulsifyingor taste-modifying agents.

The compounds of formula I and particularly A4, may be administeredorally, topically, parenterally, rectally, nasally, sublingually ortransdermally. The compound is advantageously administered by injectionor by intravenous infusion or suitable sterile solutions, or in the formof liquid or solid doses via the alimentary canal, or in the form ofcreams, ointments, patches, or similar vehicles suitable for transdermalapplications. A dose of from 0.01 μg to 1000 μg per day of the compoundsI, particularly A4, preferably from about 0.1 μg to about 500 μg perday, is appropriate for prevention and/or treatment purposes, such dosebeing adjusted according to the disease to be treated, its severity andthe response of the subject as is well understood in the art. Since thecompound exhibits specificity of action, each may be suitablyadministered alone, or together with graded doses of another activevitamin D compound—e.g. 1α-hydroxyvitamin D₂ or D₃, or1α,25-dihydroxyvitamin D₃—in situations where different degrees of bonemineral mobilization and calcium transport stimulation is found to beadvantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly A4, as defined by theabove formula I and Ia as the active ingredient, and a suitable carrier.An effective amount of such compound for use in accordance with thisinvention is from about 0.01 μg to about 1000 μg per gm of composition,preferably from about 0.1 μg to about 500 μg per gram of composition,and may be administered topically, transdermally, orally, rectally,nasally, sublingually, or parenterally in dosages of from about 0.01μg/day to about 1000 μg/day, and preferably from about 0.1 μg/day toabout 500 μg/day.

The compounds I, particularly A4, may be formulated as creams, lotions,ointments, topical patches, pills, capsules or tablets, suppositories,aerosols, or in liquid form as solutions, emulsions, dispersions, orsuspensions in pharmaceutically innocuous and acceptable solvent oroils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly A4, may be advantageously administered inamounts sufficient to effect the differentiation of promyelocytes tonormal macrophages. Dosages as described above are suitable, it beingunderstood that the amounts given are to be adjusted in accordance withthe severity of the disease, and the condition and response of thesubject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 2. The compound ofclaim 1 wherein X₃ is hydrogen.
 3. The compound of claim 1 wherein X₁ ishydrogen.
 4. The compound of claim 1 wherein X₁, X₂ and X₃ are allt-butyldimethylsilyl.
 5. A pharmaceutical composition containing aneffective amount of at least one compound as claimed in claim 1 togetherwith a pharmaceutically acceptable excipient.
 6. The pharmaceuticalcomposition of claim 5 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 7. Thepharmaceutical composition of claim 5 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 8.2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


9. A pharmaceutical composition containing an effective amount of2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ togetherwith a pharmaceutically acceptable excipient.
 10. The pharmaceuticalcomposition of claim 9 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 11. Thepharmaceutical composition of claim 9 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 12.A method of treating psoriasis comprising administering to a subjectwith psoriasis an effective amount of a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 13. The method ofclaim 12 wherein the compound is administered orally.
 14. The method ofclaim 12 wherein the compound is administered parenterally.
 15. Themethod of claim 12 wherein the compound is administered transdermally.16. The method of claim 12 wherein the compound is administeredtopically.
 17. The method of claim 12 wherein the compound isadministered rectally.
 18. The method of claim 12 wherein the compoundis administered nasally.
 19. The method of claim 12 wherein the compoundis administered sublingually.
 20. The method of claim 12 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 21. The method of claim 12 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


22. A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 23. The method ofclaim 22 wherein the compound is administered orally.
 24. The method ofclaim 22 wherein the compound is administered parenterally.
 25. Themethod of claim 22 wherein the compound is administered transdermally.26. The method of claim 22 wherein the compound is administeredrectally.
 27. The method of claim 22 wherein the compound isadministered nasally.
 28. The method of claim 22 wherein the compound isadministered sublingually.
 29. The method of claim 22 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 30. The method of claim 22 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


31. A method of treating an autoimmune disease selected from the groupconsisting of multiple sclerosis, lupus, diabetes mellitus, host versusgraft rejection, and rejection of organ transplants, comprisingadministering to a subject with said disease an effective amount of acompound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 32. The method ofclaim 31 wherein the compound is administered orally.
 33. The method ofclaim 31 wherein the compound is administered parenterally.
 34. Themethod of claim 31 wherein the compound is administered transdermally.35. The method of claim 31 wherein the compound is administered rectally36. The method of claim 31 wherein the compound is administered nasally.37. The method of claim 31 wherein the compound is administeredsublingually.
 38. The method of claim 31 wherein the compound isadministered in a dosage of from about 0.01 μg/day to about 1000 μg/day.39. The method of claim 31 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


40. A method of treating an inflammatory disease selected from the groupconsisting of rheumatoid arthritis, asthma, and inflammatory boweldiseases, comprising administering to a subject with said disease aneffective amount of a compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 41. The method ofclaim 40 wherein the compound is administered orally.
 42. The method ofclaim 40 wherein the compound is administered parenterally.
 43. Themethod of claim 40 wherein the compound is administered transdermally.44. The method of claim 40 wherein the compound is administeredrectally.
 45. The method of claim 40 wherein the compound isadministered nasally.
 46. The method of claim 40 wherein the compound isadministered sublingually.
 47. The method of claim 40 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 48. The method of claim 40 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


49. A method of treating a skin condition selected from the groupconsisting of wrinkles, lack of adequate skin firmness, lack of adequatedermal hydration and insufficient sebum secretion which comprisesadministering to a subject with said skin condition an effective amountof a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 50. The method ofclaim 49 wherein the compound is administered orally.
 51. The method ofclaim 49 wherein the compound is administered parenterally.
 52. Themethod of claim 49 wherein the compound is administered transdermally.53. The method of claim 49 wherein the compound is administeredtopically.
 54. The method of claim 49 wherein the compound isadministered rectally.
 55. The method of claim 49 wherein the compoundis administered nasally.
 56. The method of claim 49 wherein the compoundis administered sublingually.
 57. The method of claim 49 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 58. The method of claim 49 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


59. A method of treating renal osteodystrophy comprising administeringto a subject with renal osteodystrophy an effective amount of a compoundhaving the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 60. The method ofclaim 59 wherein the compound is administered orally.
 61. The method ofclaim 59 wherein the compound is administered parenterally.
 62. Themethod of claim 59 wherein the compound is administered transdermally.63. The method of claim 59 wherein the compound is administeredrectally.
 64. The method of claim 59 wherein the compound isadministered nasally.
 65. The method of claim 59 wherein the compound isadministered sublingually.
 66. The method of claim 59 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 67. The method of claim 59 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


68. A method of treating or preventing obesity of an animal, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in an animal comprising administering to an animal inneed thereof an effective amount of a compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 69. The method ofclaim 68 wherein the compound is administered orally.
 70. The method ofclaim 68 wherein the compound is administered parenterally.
 71. Themethod of claim 68 wherein the compound is administered transdermally.72. The method of claim 68 wherein the compound is administeredrectally.
 73. The method of claim 68 wherein the compound isadministered nasally.
 74. The method of claim 68 wherein the compound isadministered sublingually.
 75. The method of claim 68 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 76. The method of claim 68 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


77. The method of claim 68 wherein the animal is a human.
 78. The methodof claim 68 wherein the animal is a domestic animal.
 79. The method ofclaim 68 wherein the animal is an agricultural animal
 80. A method oftreating secondary hyperparathyroidism comprising administering to asubject with secondary hyperparathyroidism an effective amount of acompound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 81. The method ofclaim 80 wherein the compound is administered orally.
 82. The method ofclaim 80 wherein the compound is administered parenterally.
 83. Themethod of claim 80 wherein the compound is administered transdermally.84. The method of claim 80 wherein the compound is administeredrectally.
 85. The method of claim 80 wherein the compound isadministered nasally.
 86. The method of claim 80 wherein the compound isadministered sublingually.
 87. The method of claim 80 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 88. The method of claim 80 wherein the compound is2-methylene-(20S,25S)-19,26-dinor-1α,25-dihydroxyvitamin D₃ having theformula:


89. The method of claim 80 wherein the subject has chronic kidneydisease.
 90. The method of claim 80 wherein the subject is on dialysis.